Inhibitory effect of reactive oxygen species on angiotensin I-converting enzyme (kininase II)

1. Somatic angiotensin I-converting enzyme (ACE) is a protein that contains two similar domains (N- and C-terminal), each possessing an active site. W e have examined the effects of a generator of hydroxyl radicals (g(.)OH: 2, 2'-azo-bis(2-amidinopropane)) and hydrogen peroxide (H2O2) on ACE using an in vitro approach, 2. The generator of hydroxyl radicals inactivated ACE in a time (2-6 h)- an d concentration (0.3-3 mmol/L)-dependent manner at 37 degreesC, When ACE wa s coincubated for 4h with g(.)OH (3 mmol/L), its activity decreased by 70%, Addition of dimethylthiourea or mannitol + methionine, two (OH)-O-. scaven gers, resulted in a significant protection of ACE activity. Mercaptoethanol and dithiotreitol, two thiol-reducing agents, also efficiently protected A CE activity. 3. The hydrolysis of two natural and domain-specific substrates was explore d. The hydrolysis of angiotensin I, preferentially cleaved by the C-domain, was significantly inhibited (57-58%) after 4 h exposure to g(.)OH (0.3-1 m mol/L). Under the same conditions of exposure, the hydrolysis of N-acetyl-S er-Asp-Lys-Pro, a specific substrate for the N-domain, was only slightly in hibited by 1 mmol/L g . OH. 4. Hydrogen peroxide, another source of . OH, was used. After exposure to H 2O2 (3 mmol/L; 4 h), an 89% decrease in ACE activity was observed, Pretreat ment with the iron chelator deferoxamine (1 mmol/L) attenuated H2O2 mediate d ACE inactivation, demonstrating that the effect of H2O2 was partly due to its conversion into (OH)-O-. (Fenton reaction). 5. In summary, our findings demonstrate that g(.)OH and H2O2 inhibit ACE ac tivity and suggest a preferential action of g(.)OH on the C-domain of the e nzyme.